Affinity chromatography of influenza virus on immobilized alpha- and beta-ketosides of neuraminic acid derivatives

Abstract

In order to purify influenza viruses which contain neuraminidase as a constituent of the virus-coat surface, a study was made of the affinity of formaldehyde-inactivated viruses for immobilized ketosides of neruaminic acid derivatives. Attempted desorption of viruses bound to columns of the conjugates was performed by addition of the benzyl alpha-ketoside of N-acetylneuraminic acid to the eluent. The conjugates with alpha-ketosidically bound neuraminic acids adsorbed virus which could be desorbed with the enzyme substrate. When beta-ketosidically immobilized N-acetylneuraminic acid was saturated with virus and the conjugate was washed with buffer until it was free from neuraminidase activity, no more virus could be desorbed with neuraminidase substrate-containing eluents. However, addition of substrate to the buffer during the washing procedure resulted in a neuraminidase activity peak in the effluent. All conjugates bound firmly a portion of the viruses, which remained on the sorbents even after excessive treatment with eluent containing the neuraminidase substrate. When the conjugates were saturated with virus, all sites which bound the viruses strongly were blocked, and the remaining binding sites could be utilized for reversible adsorption of the viruses. Using this method, crude influenza virus vaccine could be separated into allantoic-fluid components and pure virus. Centrifugation of the virus preparation in a sucrose gradient indicated that this column procedure did not solubilize the viral neuraminidase. Using freshly prepared non-inactivated influenza virus, the portion of gel-bound viruses which could not be desorbed by neuraminidase substrate was diminished.